Fetal and umbilical blood vessels form independently; however, the means by which they connect to each other to ensure fetal-maternal exchange during gestation is obscure. The goal of this new grant proposal is to elucidate the origin and mechanism of formation of a small hemogenic vessel, provisionally named the Vessel of Confluence (VOC), in the mouse embryo. The function of the VOC is to connect the umbilical artery to the embryonic dorsal aortae, thereby ensuring a continuous arterial system between the developing conceptus and its mother. How the VOC forms is a major question in reproductive biology and essential for successful gestational outcome. However, description of the VOC has no precedent in the literature. The VOC forms in close proximity to visceral endoderm/hypoblast, an extraembryonic tissue found in all amniotes. Closer inspection of the visceral endoderm revealed the unexpected presence of a stage- and site-specific gap within an otherwise continuous epithelium. This gap separated yolk sac blood islands from allantois-associated posterior visceral endoderm (aPVE), creating within the latter a distinct developmental domain that uniquely expressed Hedgehog (Hh). Unexpectedly, the aPVE contributed descendant cells to the VOC, whose site of formation was rich in Fibroblast Growth Factor (FGF) and heparan sulfate proteoglycan (HSPG) signaling. Pharmacological inhibition of FGF and HSPG resulted in failure of the VOC to form, while alterations in Hh signaling led to significant disruptions in vessel size and shape. On the basis of these data, we hypothesize that gap formation triggers an epithelial-to-mesenchymal transition (EMT) within the aPVE, leading to formation of the VOC and orchestrated by interaction between Hh, FGFs, and HSPGs. Using the mouse as our model system, we will combine genetics and molecular biology with classical techniques of embryology in living embryos to investigate establishment of the fetal-umbilical connection. Because FGF and Hh signaling pathways play well-established critical roles in all of the fundamental biological processes under study here, including apoptosis, epithelial-to-mesenchymal transition, and vasculo/hemogenesis, and because upstream and downstream pathways are well-characterized, they are highly promising candidates in which to gain entr?e into the fetal-umbilical interface, and dissect at a hitherto unprecedented pace the complex signaling pathways that guide VOC formation. In birthing rooms around the world, the umbilical cord is just cursorily examined, scrutinized only if the fetus is in obvious distress. Yet, given te cord's intimate anatomical association with the fetal vasculature, careful analysis of the neonate's umbilical cord must undoubtedly hold clues to disease, which if identified early, promise much-improved long-term prognosis. We anticipate that successful outcome of this study will alert scientists and clinicians to the VOC as a potential risk factor in neonatal defect that merits further screening and evaluation prior to routine disposal of the afterbirth.